1. Field of the Invention
This invention relates generally to nucleic acid sequences encoding proteins that are associated with abiotic stress responses and abiotic stress tolerance in plants. In particular, this invention relates to nucleic acid sequences encoding proteins that confer drought, cold, and/or salt tolerance to plants.
2. Background Art
Abiotic environmental stresses, such as drought stress, salinity stress, heat stress, and cold stress, are major limiting factors of plant growth and productivity. Crop losses and crop yield losses of major crops such as rice, maize (corn) and wheat caused by these stresses represent a significant economic and political factor and contribute to food shortages in many underdeveloped countries.
Plants are typically exposed during their life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions of desiccation. However, if the severity and duration of the drought conditions are too great, the effects on plant development, growth and yield of most crop plants are profound. Furthermore, most of the crop plants are very susceptible to higher salt concentrations in the soil. Continuous exposure to drought and high salt causes major alterations in the plant metabolism. These great changes in metabolism ultimately lead to cell death and consequently yield losses.
Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies to develop new lines of plants that exhibit resistance (tolerance) to these types of stresses are relatively slow and require specific resistant lines for crossing with the desired line. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to drought, cold and salt tolerance in model, drought- and/or salt-tolerant plants are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways. This multi-component nature of stress tolerance has not only made breeding for tolerance largely unsuccessful, but has also limited the ability to genetically engineer stress tolerance plants using biotechnological methods.
Therefore, what is needed is the identification of the genes and proteins involved in these multi-component processes leading to stress tolerance. Elucidating the function of genes expressed in stress tolerant plants will not only advance our understanding of plant adaptation and tolerance to environmental stresses, but also may provide important information for designing new strategies for crop improvement.
One group of proteins that seems to be associated with stress responses in plants and animals are proteins related to cell division and the cell cycle. Evidence of this relationship is found in the fact that different environmental stresses related to soil water content, incident light and varying leaf temperature all result in a change in the cell division rate. For example, in sunflower leaves, there is a lengthening of the cell cycle associated with stress and aging. Cells in the sunflower leaves tend to arrest at the G1 phase of cell cycle, with no change in the duration of the S-G2-M phases of the cell cycle. Similar observations have been made in other plant species placed under environmental stresses such as sucrose starvation (Van""t Hof 1973 Bookhaven Symp. 25:152), oxidative stress (Reichheld et al. 1999 Plant J. 17:647), and water depletion (Schuppler et al. 1998 Plant Physiol. 117:667). These observations and results suggest that there is an important xe2x80x9ccheckpointxe2x80x9d in the regulation of the cell cycle at the G1-S transition, while other studies suggest another xe2x80x9ccheckpointxe2x80x9d at the G2-M transition with an arrest of the cells at the G2 phase. It has also been determined that temperature affects the duration of all cell cycle phases by a similar proportion without the preferential arrest in any particular phase of the cell cycle (Tardieu and Granier, 2000 Plant Mol. Biol. 43:555). Although these aforementioned responses to environmental stresses differ, each of these results demonstrates the interconnectivity of the stress response system of these plants and the cell division proteins therein.
The prior art describes several proteins associated with cell division and the cell cycle. One such protein, or protein complex, is the cyclin-CDK complex, which controls progression of the cell cycle phases. Cyclin-dependent protein kinases (CDKs) require cyclin binding for activity and are now widely recognized players at the checkpoints of the eukaryotic cell cycle. The widespread importance of CDKs was realized nearly ten years ago from independent genetic approaches in yeast and biochemical studies of mitotic controls in fertilized eggs of marine invertebrates.
One known component of a CDK complex is a Cdc2 protein termed P34cdc2, which is required at both control points (G1-S and G2-M). Several other Cdc2 homologs have been isolated from human and plant species including yeast. One such yeast homolog is Cdc48, which plays a role in the spindle pole body separation in Saccharomyces cerevisiae. Another Cdc2 homolog has been described in Arabidopsis (Feiler et al. 1995 EMBO J 14:5626) that is highly expressed in the proliferating cells of the vegetative shoot, root, floral inflorescence and flowers and in rapidly growing cells. The Arabidopsis Cdc48 gene is up regulated in the developing microspores and ovules and down regulated in most differentiated cell types. In addition, this gene has been localized to the nucleus and during cytokinesis to the fragmoplast.
Another group of proteins involved in cell division and the cell cycle are pRB proteins. Growing evidence suggests that pRB-like proteins in plants might be among the nuclear targets of plant CDKs. In mammals, the pRB is central to the regulation of the G1-to-S transition. Phosphorylation of mammalian pRB by cyclin D- and cyclin E-dependent kinases renders the pRB inactive and thereby represses the S phase and promotes DNA replication. Significantly, the pRB-binding motif LXCXE (where X denotes any amino acid) is found in all known plant D cyclins. In plants, LXCXE-dependent interactions between D cyclins from Arabidopsis and maize pRB proteins have been demonstrated in vitro and in a yeast two-hybrid assay. The role of pRBs in plant cell division related signaling cascades is further supported by the fact that several pRBs, and in particular, the maize pRB, contain multiple putative CDK phosphorylation sites and are efficiently phosphorylated in vitro by mammalian G1- and S-specific CDKs. Maize pRB proteins are also known to undergo changes in phosphorylation during the transition to endoreduplication in the endosperm. However, phosphorylation by plant CDKs remains to be demonstrated.
Therefore, although several plant cell cycle proteins have been elucidated, the prior art has yet to describe the plant cell cycle signal transduction cascades in detail. The prior art also fails to describe the relation between plant cell cycle proteins and a plant""s response to environmental stress such that a stress tolerant plant can be generated. Accordingly, there is a need to identify genes expressed in stress tolerant plants that have the capacity to confer stress resistance to its host plant and to other plant species. Newly generated stress tolerant plants will have many advantages, such as increasing the range that crop plants can be cultivated by, for example, decreasing the water requirements of a plant species.
This invention fulfills in part the need to identify new, unique cell cycle proteins capable of conferring stress tolerance to plants upon over-expression. The present invention provides a transgenic plant cell transformed by a Cell Cycle Stress-Related Protein (CCSRP) coding nucleic acid, wherein expression of the nucleic acid sequence in the plant cell results in increased tolerance to environmental stress as compared to a wild type variety of the plant cell. Namely, described herein are the cell cycle proteins 1) Cell Cycle-1 (CC-1); 2) Cell Cycle-2 (CC-2) and 3) Cell Cycle-3 (CC-3), all from Physcomitrella patens. 
The invention provides in some embodiments that the CCSRP and coding nucleic acid are that found in members of the genus Physcomitrella. In another preferred embodiment, the nucleic acid and protein are from a Physcomitrella patens. The invention provides that the environmental stress can be salinity, drought, temperature, metal, chemical, pathogenic and oxidative stresses, or combinations thereof. In preferred embodiments, the environmental stress can be drought or cold temperature.
The invention further provides a seed produced by a transgenic plant transformed by a CCSRP coding nucleic acid, wherein the plant is true breeding for increased tolerance to environmental stress as compared to a wild type variety of the plant. The invention further provides a seed produced by a transgenic plant expressing a CCSRP, wherein the plant is true breeding for increased tolerance to environmental stress as compared to a wild type variety of the plant.
The invention further provides an agricultural product produced by any of the below-described transgenic plants, plant parts or seeds. The invention further provides an isolated CCSRP as described below. The invention further provides an isolated CCSRP coding nucleic acid, wherein the CCSRP coding nucleic acid codes for a CCSRP as described below.
The invention further provides an isolated recombinant expression vector comprising a CCSRP coding nucleic acid as described below, wherein expression of the vector in a host cell results in increased tolerance to environmental stress as compared to a wild type variety of the host cell. The invention further provides a host cell containing the vector and a plant containing the host cell.
The invention further provides a method of producing a transgenic plant with a CCSRP coding nucleic acid, wherein expression of the nucleic acid in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant comprising: (a) transforming a plant cell with an expression vector comprising a CCSRP coding nucleic acid, and (b) generating from the plant cell a transgenic plant with an increased tolerance to environmental stress as compared to a wild type variety of the plant. In preferred embodiments, the CCSRP and CCSRP coding nucleic acid are as described below.
The present invention further provides a method of identifying a novel CCSRP, comprising (a) raising a specific antibody response to a CCSRP, or fragment thereof, as described below; (b) screening putative CCSRP material with the antibody, wherein specific binding of the antibody to the material indicates the presence of a potentially novel CCSRP; and (c) identifying from the bound material a novel CCSRP in comparison to known CCSRP. Alternatively, hybridization with nucleic acid probes as described below can be used to identify novel CCSRP nucleic acids.
The present invention also provides methods of modifying stress tolerance of a plant comprising, modifying the expression of a CCSRP in the plant, wherein the CCSRP is as described below. The invention provides that this method can be performed such that the stress tolerance is either increased or decreased. Preferably, stress tolerance is increased in a plant via increasing expression of a CCSRP.